Adhesion of the blood-borne monocyte to the endothelium is the initial event in the extravasation of this leukocyte into the vessel wall or the interstitial space. The long term objectives of this proposal are (1) to characterize biochemically the cell surface molecules on the endothelium which are involved in monocyte-endothelial cell (EC) attachment, and (2) to determine the mechanism of focal adhesion of monocytes to the vessel wall in response to hypercholesterolemia. These objectives will be pursued principally by a series of in vitro experiments utilizing a model system which involves cultured EC and the human monocytic tumor cell line U937. Experiments will be repeated where appropriate with purified blood monocytes. Our working hypothesis is that EC, in response to certain physiological stimuli, express specific cell surface receptors or binding sites for monocytes. Further, we hypothesize that migrating and/or proliferating EC express an increased number of monocyte binding sites and that this phenomenon is responsible for the focality of monocyte binding in vivo. Finally, we hypothesize that by blocking the initial adhesion of monocytes to the endothelium during the initial stages of hypercholesterolemia that subsequent events, such as fatty streak development, will not occur. Our hypotheses will be tested by pursuit of the following specific aims: 1) To characterize and isolate the binding site(s) for monocytes on the EC surface. This aim includes studies on a) the protease, glycosidase and lectin sensitivity of monocyte-EC attachment and b) studies on the detergent solubilization and purification of the EC surface molecule(s) involved in the cell-cell adhesion. 2) To prepare serum antibodies and/or monoclonal antibodies to EC surface moieties involved in monocyte-EC attachment. These antibodies will be useful in purification, in the localization and quantification of monocyte binding sites in vivo, and in the blocking of monocyte attachment to the endothelium in vivo (Aim 5). 3) To identify culture conditions or exogenous agents which regulate the expression of monocyte binding sites on the EC surface. We will examine the role of a) time in culture, b) EC migration vs. proliferation, c) EC "injury" agents and d) hypercholesterolemic vs. normal serum, in the ability of EC to bind monocytes. 4) To compare monocyte attachment to EC from hypercholesterolemic vs. normal animals and to compare monocyte binding to cultured EC from large vessels and small vessels. 5) To inhibit monocyte attachment to the pig or rabbit aorta in vivo by the infusion of antibodies against the monocyte binding site on cultured EC. By gaining mechanistic knowledge on the molecular level of this single event in monocyte-vessel wall interactions, we may be one step closer to preventing pathological phenomena that require monocyte infiltration.